Novel pyrazole-3-carboxamide derivatives as cannabinoid-1 (CB1) antagonists: Journey from non-polar to polar amides

Article abstract of DOI:10.1016/j.bmcl.2010.10.055

The synthesis and biological evaluation of novel pyrazole-3-carboxamide derivatives as CB1 antagonists are described. As a part of eastern amide SAR, various chemically diverse motifs were introduced. In general, a range of modifications were well tolerat

Full text of DOI:10.1016/j.bmcl.2010.10.055

Bioorganic & Medicinal Chemistry Letters 21 (2011) 562–568
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrazole-3-carboxamide derivatives as cannabinoid-1 (CB1)
antagonists: Journey from non-polar to polar amides q
⇑
Pradip K. Sasmal , D. Srinivasa Reddy, Rashmi Talwar, B. Venkatesham, D. Balasubrahmanyam, M. Kannan,
P. Srinivas, K. Shiva Kumar, B. Neelima Devi, Vikram P. Jadhav, Sanjoy K. Khan, Priya Mohan,
Hira Chaudhury, Debnath Bhuniya, Javed Iqbal, Ranjan Chakrabarti
Discovery Research, Dr. Reddy’s Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500049, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of novel pyrazole-3-carboxamide derivatives as CB1 antagonists
are described. As a part of eastern amide SAR, various chemically diverse motifs were introduced. In
general, a range of modifications were well tolerated. Several molecules with high polar surface area were
also indentified as potent CB1 receptor antagonists. The in vivo proof of principle for weight loss is
exemplified with a lead compound from this series.
Received 30 August 2010
Revised 12 October 2010
Accepted 13 October 2010
Available online 27 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid-1 (CB1) receptor
CB1 antagonists
Pyrazole-3-carboxamides
Obesity
DIO mice model
The prevalence of obesity is rapidly increasing globally. Obesity
has reached epidemic proportions in both developed and developing
countries. Although obesity is associated with the pathogenesis of
major diseases including diabetes or cardiovascular diseases, no
satisfactorily safe and effective obesity drugs are available at the
moment. Thus, there is a tremendous unmet need to treat obesity
and make a significant impact on the lives of the obese.
nitive disorders,15 septic shock,15 psychosis,15,16 addiction,17 and
gastrointestinal disorders.18 Another cannabinoid receptor, CB2 is
related to immune regulation and neurodegeneration.19 Therefore,
the CB2/CB1 selectivity should be taken into consideration for new
drug development of antiobesity agent. Significant efforts around
the globe led to the identification of several potent and selective
CB1 antagonists which were tested in clinical trials.
The endocannabinoid system (ECS), and specifically the
cannabinoid type 1 (CB1) receptor, plays a pivotal role in energy
homeostasis.1–3 As such, stimulation of the ECS promotes food
intake and energy storage and may be chronically overactive in
obese subjects.4–7 CB1 receptor-deficient mice are resistant to
diet-induced obesity even though their total caloric intake is similar
to that of wild-type littermates.8 In contrast, blockade of the CB1
receptor in the central nervous system decreases food intake and
increases energy expenditure, leading to a reduction in body
weight.9–12 Such an approach is particularly interesting since it not
only causes weight loss but also reverses the metabolic effects of
obesity such as insulin resistance and hyperlipidemia.13 Besides
obesity, blocking CB1 receptor may have potential in the treatment
of a number of diseases such as neuroinflammatory disorders,14 cog-
Unfortunately, due to various psychiatric events in human trials
and regulatory hurdles several CB1 receptor inverse agonists/
antagonists were recently withdrawn from clinical development
including rimonabant (1)20 (Fig. 1), taranabant (2),21 otenabant
(3),22 and ibipinabant (4).23 However, despite consecutive failures
of leading CB1 receptor antagonists, works continue to identify
novel peripherally restricted CB1 antagonists24 that limit BBB pen-
etration so that they do not induce serious psychiatric disorders.
One possibility to achieve this would be by making compounds
having considerably higher polar surface area (PSA) and lower
lipophilicity. In considering options for designing agents that
would potentially have an improved profile relative to 1, one of
the elements we chose to replace was the hydrazide functionality
with an amide. Subsequently, polar motifs were introduced to
reduce CNS exposure. We described herein our efforts to develop
proprietary series with desirable physicochemical properties by
focusing SAR of eastern amide zone and subsequent modification
in other parts to identify lead compounds.
q
DRL Publication No. 723.
⇑
Corresponding author at present address: Dr. Reddy’s Laboratories Ltd,
Innovation Plaza, Bachupally, Hyderabad 500072, India. Tel.: +91 40 4434 6865;
fax. +91 40 4434 6125.
The target compounds (Tables 1–5) were synthesized as
outlined in Schemes 1–8. The synthesis of the core acids IV were
E-mail address: pradipks@drreddys.com (P.K. Sasmal).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.055

P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
563
Table 2
Rat CB1 binding affinities of compounds 13–29
O
N
N
O
R
NH
O
O
N
NC
Cl
N
H
NH
Ar1 = 4-chlorophenyl
CF3
N
N
Ar2 = 2,4-dichlorophenyl
Ar1
N
Cl
Cl
2
(taranabant)
Ar2
Compd
R
tPSAa
57.1
rCB1_IC50 (nM)
2
b
Cl
1
(rimonabant)
13
N
N
O
S
NH
O
O
MeN
N
14
15
69.4
56.7
57
20
NH2
Cl
N
N
N
NHEt
N
N
H
N
N
N
Cl
Cl
N
N
16
17
60.3
60.3
71
20
N
Cl
N
3 (otenabant)
4 (ibipinabant)
N
N
Figure 1. Structures of CB1 antagonists.
18
19
72.7
72.7
15
N
N
N
N
16
Table 1
c
Rat CB1 binding affinities of compounds 5–12
20
21
47.9
47.9
N
N
N
N
N
O
R
455
47
Ar1 = 4-chlorophenyl
N
Ar1
O
Ar2 = 2,4-dichlorophenyl
N
22
23
24
57.2
60.0
51.2
Ar2
NH
N
Compd
tPSAa
35.9
rCB1_IC50 (nM)
18
b
900
310
R
5
N
Et
N
6
7
35.9
35.9
20
1
25
51.2
855
N
N
N
N
iPr
N
N
N
N
26
27
28
29
51.2
68.3
68.3
77.5
>1000d
633
N
N
N
N
COMe
8
35.9
96
SO2Me
CO2Me
980
9
10
11
44.7
44.7
44.7
0.6
10
26
NH
NH
29
a
b
and as in Table 1.
50% binding at 1
34% binding at 1
c
l
l
M.
M.
NH
NH
d
12
44.7
21
routine transformations furnished compounds 24–29 as outlined
in Scheme 2.
a
tPSA: topological polar surface area.
b
The synthesis of compounds 30–36 is outlined in Scheme 3. The
coupling of IVa with 3-amino-3-methylbutanenitrile produced
intermediate amide VI. The cyano moiety was converted to various
functional groups by standard protocols generating compounds
30–32 and the tetrazole 35 which after methylation under
MeI/K2CO3 conditions in DMF afforded compounds 36 as the major
product.25 On the other hand coupling of IVa with 2-amino-2-
methylpropan-1-ol and 3-amino-3-methylbutan-1-ol furnished
compounds 33 and 34, respectively.
values are mean of at least two experiments.
achieved following Scheme 1 from the corresponding propiophe-
none or 1-phenylbutan-1-one derivatives I.
Compounds 5–22 were synthesized by coupling of 5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbox-
ylic acid (IVa) with various primary and secondary amines under
EDC/HOBt conditions (Scheme 2). The amide coupling of tert-butyl
4-(2-amino-2-methylpropyl)piperazine-1-carboxylate with the
same acid produced intermediate V. Deprotection of the Boc group
within V with TFA afforded compound 23 which after various
Compound 37 was synthesized following Scheme 4. Ring open-
ing of the sulfamate derivative VII26 with 5-methyl-2H-tetrazole
followed by debenzylation gave 2-methyl-1-(5-methyl-2H-tetra-

564
P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
Table 3
Table 5
Rat CB1 binding affinities of compounds 30–41
Rat CB1 binding affinities of compounds 51–60
N
N
O
N
N
N
O
R
R1
NH
NH
Ar1 = 4-chlorophenyl
N
N
Ar1
Ar2 = 2,4-dichlorophenyl
R2
N
Cl
Ar2
b
Compd
R
tPSAa
71.0
rCB1_IC50 (nM)
Cl
O
Compd
R
R2
tPSAa
rCB1_IC50 (nM)
b
30
17
O
51
52
53
54
55
56
57
58
59
60
Et
Et
Me
Me
Cl
Br
85.0
85.0
39
4
O
O
31
32
82.0
87.8
>1000c
25
OSO2Pr
CN
128.4
108.8
108.8
105.3
85.0
122.3
111.3
148.6
7
31
34
8
OH
Et
CN
Cl
Cl
Cl
CH2OH
CH2F
CO2H
CO2Et
CH2OH
NH2
3
OH
>1000c
1
33
34
64.9
64.9
60
33
Cl
OH
N
OSO2Pr
0.5
N
N
>1000d
71
a
b
35
36
37
NH
93.8
85.0
85.0
and as in Table 1.
N
N
c
45% binding at 1
l
M.
N
N
N
Li
N
N
O
O
O
18
N
N
a
Y
CO2Et
N
I
II
N
X
38
105.3
102.1
74
6
X
Y
N
NHNH2
·HCl
Cl
OH
X = Cl, Br, CN, OSO2Pr
Y = H, Me
N
N
b
N
Cl
39
40
N
O
O
O
N
Y
OH
Y
OEt
N
85.0
85.0
11
88
N
N
c
N
N
N
N
N
X
X
N
41
Cl
Cl
N
N
III
IV
a
b
and as in Table 1.
c
20% binding at 1
18% binding at 1
l
l
M.
M.
Cl
Cl
d
Scheme 1. Reagents and conditions: (a) LiHMDS, ether, À78 °C, (CO2Et)2, rt, 14 h
(for Y = H); LiHMDS, tBuOMe, À20 °C, ImCOCO2Et, rt, 14 h (for Y = Me); (b) (i) EtOH,
reflux, 16 h; (ii) HOAc, reflux, 12 h; (c) LiOH, EtOH–H2O, 1–6 h.
Table 4
Rat CB1 binding affinities of compounds 41–50
Boc
O
N
R2
R1
N
N
Boc
N
N
O
N
OH
+
N
a
NH
N
N
N
Ar1 = 4-chlorophenyl
Ar2 = 2,4-dichlorophenyl
O
N
H2N
Ar1
N
Ar1
N
H
N
Ar2
Ar1 = 4-chlorophenyl
Ar2
Ar2 = 2,4-dichlorophenyl
IVa
Ar1
V
N
b
Compd
R1
R2
tPSAa
rCB1_IC50 (nM)
Ar2
R'
41
42
43
44
45
46
47
48
Me
H
Me
H
85.0
85.0
85.0
85.0
85.0
85.0
105.3
85.0
88
100
61
35
10
18
77
16
HN
R"
a
b
27
d
–CH2CH2–
Et
Et
23
–CH2CH2CH2–
e
5-22
Me
Me
Et
–CH2OH
28
29
c
f
–(CH2CH2)2
24-26
49
85.0
97.1
14
c
Scheme 2. Reagents and conditions: (a) amines, EDC, HOBt, Et3N, CH2Cl2, rt, 4–
10 h; (b) TFA, CH2Cl2, rt, 1 h; (c) NaH, DMF, alkyl iodide, 0 °C, 1 h; (d) AcCl, Et3N,
CH2Cl2, 0 °C to rt, 1 h; (e) MsCl, Et3N, CH2Cl2, 0 °C to rt, 30 min; (f) ClCO2Me, Na2CO3,
THF, rt, 16 h.
50
–(CH2CH2)2NH
a
b
and as in Table 1.
c
50% binding at 1 lM.

P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
565
CN
O
O
N
CN
a
a
b
CN
Y
N
H
51-55
N
IV
+
H2N
IVa
+
CN
H2N
H
N
XI
Ar1
N
N
X
Cl
OH
n
a
Ar2
H2N
d
X = Cl, Br, CN, OSO2Pr
Y = H, Me
32
35
VI
Cl
OH
n
b
O
N
e
c
d
c
N
H
41
56
57
53
60
30
e
Ar1
N
Ar2
f
c
f
58
59
33
: n = 1
36
31
Scheme 8. Reagents and conditions: (a) EDC, HOBt, Et3N, CH2Cl2, rt, 4–10 h; (b) (i)
NaN3, DMF, NH4Cl, 120 °C, 16 h; (ii) MeI, K2CO3, acetone, rt, 14 h; (c) (i) NBS, CCl4,
(PhCO)2O2, reflux, 16 h; (ii) AgNO3, 70% aqueous acetone, reflux, 14 h; (d) DAST,
CH2Cl2, rt, 16 h; (e) (i) PCC, CH2Cl2, rt, 14 h; (ii) NaClO2, NaH2PO4, 2-methyl-2-
butene, tBuOH/THF/H2O (4:2:1), rt, 1 h; (f) SOCl2, CHCl3, reflux, 1 h; (ii) EtOH, rt,
16 h.
34: n = 2
Scheme 3. Reagents and conditions: (a) EDC, HOBt, Et3N, CH2Cl2, rt, 10 h; (b) dry
HCl, MeOH, rt, 24 h; (c) LiOH, MeOH/THF/H2O, rt, 1 h; (d) TFA/H2SO4 (4:1), rt,
30 min; (e) NaN3, DMF, NH4Cl, 120 °C, 16 h; (f) MeI, K2CO3, acetone, rt, 14 h.
O
O
O
zol-2-yl)propan-2-amine which after coupling with IVa afforded
the desired compound.
N
N
S
Bn
N
HN
N
a
a
N
IVa
+
N
+
37
H2N
N
Compounds 38 and 39 were synthesized following protocols as
outlined in Scheme 5. The Swern oxidation of 33 followed by
treatment of the resulting aldehyde with KCN furnished the
cyanohydrin intermediate VIII. Further manipulation viz.,
(i) protection of the hydroxyl group as its acetate, (ii) tetrazole
formation, (iii) N-methylation, and (iv) deacetylation furnished
the desired compound 38. PCC mediated oxidation of 38 afforded
keto tetrazole derivative 39.
The synthesis of compounds 40–49 were accomplished from IVa
and various cyano amines by standard protocols as illustrated in
Scheme 6. The synthesis of compound 50 is outlined in Scheme 7.
The aminocyanation reaction on tert-butyl 4-oxopiperidine-1-
carboxylate produced tert-butyl 4-amino-4-cyanopiperidine-1-
carboxylate which was coupled with IVa to afford intermediate X.
The cyano group was converted to the corresponding tetrazole
which after N-methylation and deprotection of N-Boc furnished
the piperidine appended tetrazole derivative 50.
N
VII
Scheme 4. Reagents and conditions: (a) (i) Cs2CO3, DMF, 60 °C, 3 h; (ii) 10% Pd/C,
HCO2NH4, MeOH, reflux, 14 h; (b) EDC, HOBt, Et3N, CH2Cl2, rt, 10 h.
CN
O
a
N
b
c
OH
33
38
39
H
N
Ar1
N
Ar2
VIII
Scheme 5. Reagents and conditions: (a) (i) (COCl)2, DMSO, Et3N, CH2Cl2, À78 °C to
rt, 2 h; (ii) KCN, THF/H2O, rt, 1 h; (b) (i) Ac2O, Py, CH2Cl2, 0 °C to rt, 16 h; (ii) NaN3,
DMF, NH4Cl, 120 °C, 16 h; (iii) MeI, K2CO3, acetone, rt, 4 h; (iv) 1 M aqueous K2CO3,
MeOH, rt, 16 h; (c) PCC, CH2Cl2, rt, 24 h.
The coupling of 2-amino-2-methylpropanenitrile with various
substituted core acids IV and subsequent manipulation produced
compounds 51–55 as described in Scheme 8. Compounds 56 and
60 were synthesized from 41 and 53 respectively by photochemi-
cal bromination followed by AgNO3 mediated hydrolysis. Reaction
of DAST with 56 furnished the corresponding fluoromethyl analog
57. The hydroxymethyl group within 56 was converted to the
carboxylic acid functionality (58) by a two-step oxidation protocol
(PCC and NaClO2). A simple esterification led to the formation of
compound 59.
R2
R1
CN
n
R1
R2
O
N
a
b
CN
n
N
H
40
IVa
+
H2N
n = 2
IX
n = 0, 2
Ar1
b
N
41-49
Ar2
n = 0
Scheme 6. Reagents and conditions: (a) EDC, HOBt, Et3N, CH2Cl2, rt, 12 h; (b) (i)
NaN3, DMF, NH4Cl, 120 °C, 16 h; (ii) MeI, K2CO3, acetone, rt, 4 h.
The target compounds were evaluated in vitro in a rat CB1 bind-
ing assay27 and results are shown in Tables 1–5. Select compounds
were then tested for hCB2 receptor affinity studies (Table 6).27
Initially the hydrazide moiety of 1 was replaced with an amide
of tetrahydroisoquinoline (compound 5 in Table 1) which showed
good rCB1 binding affinity with IC50 18 nM. To introduce novelty
into this template mono and gem-dimethyl groups were intro-
duced. As mentioned in Table 1, compound 7 with a gem-dimethyl
group showed superior potency over 5 and 6. Cleaving the tetrahy-
droisoquinoline ring system to compound 8 showed 96-fold loss in
potency. The phentermine amide 9 showed gratifyingly very high
potency of IC50 0.6 nM. Both 7 and 9 showed good selectivity pro-
file against hCB2 as well (Table 6). Pulling out gem-dimethyl group
from 9 ended up with 16-fold loss in potency as mentioned for
compound 10. Replacing phenyl group (compound 11) with a
Boc
N
Boc
N
Boc
N
a
b
c
O
N
50
CN
N
H
CN
H2N
O
X
Ar1
N
Ar2
Scheme 7. Reagents and conditions: (a) NaCN, NH4Cl, NH4OH, rt, 24 h; (b) IVa, EDC,
HOBt, Et3N, CH2Cl2, rt, 14 h; (c) (i) NaN3, DMF, NH4Cl, 120 °C, 16 h; (ii) MeI, K2CO3,
acetone, rt, 4 h; (iii) TFA, CH2Cl2, rt, 2 h.

566
P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
Table 6
At this juncture we observed that most of the potent
compounds showed poor metabolic stability.29 Metabolite identifi-
cation work indicated that methylene group flanked between
gem-dimethyl center and heterocycles is the hot spot. We adopted
three strategies viz., (i) introduction of additional methylene unit,
(ii) oxidation of the methylene center, and (iii) removal of the
hot spot. The insertion of additional methylene as in compound
40 gave 11 nM binding potency against rCB1. The oxidation of
methylene to its hydroxyl analog as in 38 showed equipotency to
36 whereas the corresponding carbonyl analog 39 with PSA of
102.1 displayed very good rCB1 potency of 6 nM. Removing the
methylene unit as in 41 showed moderate potency.
Selectivity profile of CB1 ligands against hCB2
Compd
rCB1_IC50 (nM)
hCB2 affinity IC50 (nM)
or% binding @1 lM
IC50’s ratio
hCB2/rCB1
7
9
1
0.6
2
800
149
800
248
—
260
—
32
33
139
78
41
—
3.8
—
—
—
—
13
17
29
31
36
37
38
39
40
41
45
46
48
49
51
52
53
54
56
57
59
60
71%
5200
70%
790
2350
2500
2900
248
35%
338
55%
100
75%
67%
77%
165
1040
1020
5000
1010
30%
46%
20
29
25
71
18
74
6
11
88
10
18
16
14
39
4
Both 39 and 41 showed very good metabolic stability30 but the
selectivity against hCB2 was not high enough for these molecules.
We next investigated the impact of replacing gem-dimethyl group
with other moieties in CB1 potency and selectivity and the results
are summarized in Table 4. In few occasion, potency was retained
and moderate as in case of 41 whereas molecules like 45, 46, 48,
and 49 showed 5–8-fold improvement in CB1 potency but didn’t
impact much in terms of selectivity. The piperidine analog 50 com-
pletely lost its potency.
To introduce additional polarity into the template of 41, further
modification was done in the 4-methyl position of pyrazole as well
in the chlorophenyl region and the results are summarized in
Table 5. Most of these modifications were well tolerated with im-
proved selectivity profile. Compound 53 having propane sulfonate
functionality with PSA of 128.4 displayed potency of 7 nM for CB1
and 149-fold selectivity against CB2 whereas the corresponding
cyano analog 54 showed CB1 potency of 31 nM with moderate
selectivity. Replacement of the methyl group with more polar
hydroxymethyl functionality as in compound 56 with PSA of
105.3 displayed good rCB1 binding affinity (IC50 8 nM) with
625-fold selectivity against hCB2. The corresponding fluoro analog
57 was also potent displaying 3 nM potency. Replacement of the
methyl group with carbethoxy group as in 59 displayed 1 nM
potency whereas the corresponding carboxylic acid 58 was
completely detrimental to CB1. Combining the features of 53 and
56 led to the identification of very polar compound 60 (PSA
148.6) which displayed very high CB1 potency (IC50 0.5 nM) with
very high selectivity of >1000-fold. All these potent and selective
CB1 ligands also showed good metabolic stabilities.30
As discussed above a number of compounds with low nanomo-
lar binding affinity for CB1 with good selectivity profile against CB2
have been identified. Select compounds were initially assessed for
its central pharmacodynamic effect in the hypothermia model31,32
and the results are summarized in Table 7. All of these compounds
have shown moderate to good inhibition of CB1 agonist
(WIN-55212) induced hypothermia in SAM model. One of the lead
compounds 56 has shown dose dependant reduction in acute food
intake (Fig. 2)33 when tested at 3, 10, and 30 mg/kg po in SAM
model. The effect of rimonabant at 10 mg/kg po was comparable
to the highest dose of 30 mg/kg po of compound 56 suggesting that
rimonabant might be having more pronounced central effect
—
41
149
33
625
337
—
7
31
8
3
1
0.5
—
cyclohexyl group showed loss in potency. The cyclopentyl counter-
part displayed a similar result. But all of these compounds are suf-
ficiently non-polar with lower PSA.28
After establishing good CB1 potency with proprietary amide
motif within rimonabant template, attention was shifted to replace
lipophilic eastern part with various heterocyclic groups and the re-
sults are summarized in Table 2. The 2-pyridyl analog (13) showed
2 nM potency whereas 2-pyrazinyl and 3-indolyl moieties (14 and
15) showed loss in potency. The compound with 1-imidazolyl moi-
ety displayed significant loss in potency whereas the correspond-
ing 4-methyl analog showed improvement in rCB1 potency (16
vs 17) as well as selectivity of 260-fold against hCB2. Both the iso-
meric triazoles 18 and 19 were tolerated displaying similar poten-
cies. Replacement of five-membered hetero-aryls with pyrrolidine
(20) was detrimental to CB1 potency whereas little improvement
in potency was observed with piperidine moiety (21) though the
corresponding morpholine analog (22) was well tolerated showing
rCB1 binding affinity of 47 nM. The piperazine moiety was not tol-
erated whereas CB1 potency was improved with the corresponding
N-methyl piperazine analog (24). These observations showed less
tolerance for HBD in this region. Replacement of N-methyl with
bulkier ethyl and iso-propyl groups (24 vs 25 and 26) were detri-
mental to CB1 potency.
Though the acetyl and mesylate analogs were not tolerated the
corresponding carbamate (29) displayed good potency (IC50
29 nM) in rCB1 binding assay. This observation encouraged us to
explore the tolerance of carboxylate functionality in this region.
When the methyl piperazine-1-carboxylate was replaced with a
much shorter group, methyl carboxylate, compound 30 showed
good potency of 17 nM in rCB1 binding assay though the carboxylic
acid 32 was completely detrimental. More polar carboxamide
derivative 31 was also equipotent to ester displaying IC50 of
25 nM. The hydroxyl functionality with one and two methylene
units as in 33 and 34 also retained their potencies. The in vitro
potency of 30 encouraged us to explore the bioisosteric and more
polar tetrazole motif. The N-methyl tetrazole 36 having PSA of
85.0 showed acceptable potency whereas the N-unsubstituted tet-
razole (35), like its carboxylic acid counterpart lost all its potency.
The isomeric tetrazole 37 displayed fourfold increase in rCB1
potency.
Table 7
Inhibition of CB1 agonist (WIN-55212) induced hypothermia in Swiss Albino Mice
(SAM)
Compd % Inhibition of WIN-55212 induced hypothermia at 30 min in SAM
3 mpk po
10 mpk po
31
39
41
52
53
56
60
—
94
—
54
89
—
84
—
70
—
—
95
48
—

P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
567
Supplementary data
Supplementary data (synthetic procedures and characterization
data of all 56 compounds 5–60) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.10.055.
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Figure 3. Effect on body weight after subchronic oral administration of 56
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25. A 2:1 mixture of N2- and N1-methylated products were formed which were
easily separable by flash chromatography. In proton NMR, the CH3 signal of N2-
methylated product always appears in downfield compared to the CH3 signal
of N1-methylated product. The structure of 36 was also confirmed by NOE
experiments.
eral,
a range of modifications were well tolerated. Several
molecules with high polar surface area were also indentified as
potent CB1 antagonists. The representative molecule 56 showed
significant anti-obesity effect in a DIO mice model after chronic
treatment for 15 days. The most polar compound 60 displaying
sub-nanomolar potency for CB1 requires additional experimenta-
tion to demonstrate its peripheral mode of action. Besides obesity,
the disclosed CB1 ligands might find their application in pharma-
cological intervention of other diseases involving CB1 signaling
pathways.35
26. Posakony, J. P.; Grierson, J. R.; Tewson, T. J. J. Org. Chem. 2002, 67, 5164.
27. Assay protocols for in vitro binding affinities against rCB1 and hCB2: The
molecules were tested for potential binding to CB1 receptor using rat brain
Acknowledgments
extract and [3H]-SR141716A. Briefly, membranes (ꢀ5
lg) were incubated at
30 °C with [3H]-SR141716A (2 nM) in 0.25 ml of Tris based buffer, pH 7.4 for
We thank the analytical department of Discovery Research for
providing characterization data of all compounds.
1 h. A rapid filtration technique using Whatman GF/C filters [pretreated with

568
P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 562–568
0.5% (v/v) Brij-35], and a 96-well filtration apparatus was used to harvest and
the second test. The vehicle tests were done to acclimatize the animals to
experimental conditions. Twenty four hours after the second vehicle test,
animals were kept for fasting for 24 h. On the day of the experiment, the
animals were housed in individual cages and dosed with test compounds/
vehicle. After the first hour, a measured amount of food was made available
and the amount of food consumed within the first hour was recorded.
34. All animals (c57B/L6 mice) were maintained on 12 h light–dark cycle at 25 °C.
They were given high fat diet (Research Diets, D12492, 60% kcal fat) for
3 months to develop obesity and randomized to control and treatment groups
based on their body weight. Test compound(s) were administered through oral
gavage to the animals, once daily. Control animals received vehicle (0.25%
CMC) only. The animals were maintained on same high fat diet throughout the
study. Body weight and food consumption were monitored daily for the first
7 days and subsequently every 3 days.
35. (a) Turcotte, D.; Le Dorze, J.-A.; Esfahani, F.; Frost, E.; Gomori, A.; Namaka, M.
Expert Opin. Pharmacother. 2010, 11, 17; (b) Parolaro, D.; Realini, N.; Vigano, D.;
Guidali, C.; Rubino, T. Exp. Neurol. 2010, 224, 3; (c) Ligresti, A.; Petrosino, S.; Di
Marzo, V. Curr. Opin. Chem. Biol. 2009, 13, 321; (d) Bermudez-Silva, F. J.;
Viveros, M. P.; McPartland, J. M.; Rodriguez de Fonseca, F. Pharmacol. Biochem.
Behav. 2010, 95, 375; (e) Bosier, B.; Muccioli, G. G.; Hermans, E.; Lambert, D. M.
Biochem. Pharmacol. 2010, 80, 1.
rinse labeled membranes (six times with 0.5 ml of cold buffer containing 0.25%
bovine serum albumin). The radioactivity bound to the filters was counted
with biofluor liquid scintillant. Non specific binding was determined in
presence of 10 lM of unlabeled ligand. A similar assay was conducted with
membrane preparations derived from hCB2-CHO cells for analyzing binding to
CB2 receptors using [3H]-CP 55,940 as the input ligand.
28. tPSA was calculated using ChemBioDraw Ultra 11.0 software.
29. Metabolic stability <20% after 30 min when test compounds incubated in mice
and rat liver microsomes.
30. Metabolic stability >60% after 30 min.
31. CB1 agonists induce
a reduction in body temperature by acting on CB1
receptors in the hypothalamus, an effect that can be reversed by an antagonist
32. Protocol for hypothermia studies: animals were grouped based on body weight
and basal rectal temperatures were measured. They were then dosed orally
with either test compounds or vehicle and 1 h later the rectal temperatures
were recorded again. 2 mg/kg of Win 55212-2 was then injected through the
tail vein and the rectal temperatures were measured 0.5 h post injection.
33. Protocol for acute food intake studies: The acute effects of the drug on food
consumption were analyzed in animals deprived of food for 24 h and
habituated to handling. Prior to the start of the experiment, animals were
subjected to two vehicle tests and grouped based on their food consumption in